Major traumatic life events are risk factors for stress-related neuropsychiatric disorders, often accompanied by systemic inflammation, neuroinflammation, and microglial priming. As systemic inflammation, neuroinflammation, and microglial priming are considered risk factors for developing stress-related psychiatric disorders, one novel therapeutic strategy is to identify interventions that mitigate these responses. In this study, we investigated the effects of a novel soil-derived Mycolicibacterium, Mycolicibacterium sp. strain KGA-10, on in vitro immunoregulatory potential in murine bone marrow-derived dendritic cells (BMDCs) and on biomarkers of systemic inflammation, biomarkers of hippocampal neuroinflammation and microglial priming, and anxiety-like defensive behavioral responses in adult male rats exposed to inescapable tail-shock stress (IS). In Experiments 1 and 2, BMDCs were exposed to the type strain, Mycolicibacterium vaccae ATCC 15483 (0, 10, 30, 100, 300 µg/mL; Experiment 1) or M. sp. strain KGA-10 (100 µg/mL; Experiment 2) or sterile borate-buffered saline (BBS) vehicle followed, 24 h later, by exposure to lipopolysaccharide (LPS; 250 ng/mL) or a cell culture media vehicle, then, 24 h later, assessed for Il10, Il12a, and Il12b mRNA expression. Exposure of murine BMDCs to M. vaccae ATCC 15483 or M. sp. strain KGA-10 induced an immunoregulatory phenotype, characterized by increased ratios of Il10:Il12a and Il10:Il12b mRNA expression in both naïve and lipopolysaccharide- (LPS; 250 ng/mL) challenged conditions. In Experiment 3, adult male rats received weekly injections of heat-killed M. sp. strain KGA-10 (0.1 mg/0.1 mL, s.c.) or sterile BBS vehicle over three weeks prior to IS. Anxiety-like defensive behavioral responses were assessed 24 h following IS or home cage control conditions using the juvenile social exploration (JSE) test, while biomarkers of hippocampal neuroinflammation and microglial priming were assessed using real-time reverse transcription - polymerase chain reaction (real-time RT-PCR). M. sp. strain KGA-10 treatment promoted an anti-inflammatory immunophenotype, evidenced by decreased hippocampal Il12a, and decreased biomarkers of microglial priming, Nfkbia and Nlrp3 mRNA expression among rats exposed to IS, in association with prevention of IS-induced increases in anxiety-like defensive responses in the JSE test. These findings suggest that M. sp. strain KGA-10 is a promising candidate for a novel intervention for promotion of stress resilience and prevention of stress-related psychiatric disorders.

INTRODUCTION:Military personnel are exposed to airborne hazards, such as jet fuel, that may lead to adverse health outcomes that come from biological mechanisms. In the serum of healthy individuals, the levels of microRNA are fairly consistent over time; therefore, alterations in the expression levels of specific miRNA may reflect the effects of environmental or occupational exposures on epigenetic mechanisms. The United States established a biorepository for storing serum samples collected from military members over the lifetime of their active duty (Department of Defense Serum Repository [DODSR]); however, samples may be decades old and are not stored in tubes meant to preserve RNA. The purpose of this study was to develop optimized miRNA extraction protocols for downstream RNA sequencing using DODSR samples as a proof of concept, with potential future applicability to other biorepositories. In addition, the optimized protocol was used to process and sequence over 1,000 DODSR samples. MATERIALS AND METHODS:Seven optimization experiments were conducted using existing serum samples that were collected and frozen at -80 °C for up to 2 years prior to experiments. After optimizing the processing method, a total of 1,016 samples from the DODSR were processed as proof of concept. miRNAs were extracted, and quality control measures were assessed via the TapeStation system or paired-end sequencing. RESULTS:We achieved successful miRNA sequencing for over 86% of samples that were, on average, collected more than 16 years prior. Furthermore, our analysis revealed a significant correlation between the ability to sequence serum samples and the age of the sample. Results from optimizing seven different processes suggested that incorporating spike RNA and tightly controlled sample thaws using an aluminum block are essential for high-quality sequence outcomes. Low concentration of miRNA in stored serum presents unique challenges for using standard quality control metrics. CONCLUSIONS:Overall, this study established protocols for measuring miRNA in DODSR samples for studies that aim to elucidate early detection biomarkers or mechanistic associations between occupation-related toxic exposures and negative health outcomes.

A combination of multiple factors (e.g., deployment tempo, toxic exposures, smoking, stress) has resulted in increased prevalence of Veterans with chronic respiratory health conditions. Concurrently, researchers and clinicians have observed respiratory health conditions and mental health outcomes are pervasive in Veterans with a history of post 9-11 deployments, with 40% increase in those with respiratory illness also diagnosed with depression. New onset of airborne pollutants can trigger biological response through inflammation and irritation that may limit pharmacological (e.g., anti-inflammatory medication) and interventional treatment (e.g. behavior activation therapy) efficacy. The objective of this one arm pilot study was to establish the acceptability and feasibility of a portable air cleaner (PAC) in a population of Veterans diagnosed with chronic respiratory health conditions and major depressive disorder (MDD). To accomplish this goal, portable air cleaners and indoor air quality measurement devices were placed in the residence of Veterans with clinically diagnosed chronic respiratory health conditions and MDD. The primary outcome of this study was feasibility and acceptability survey results. In addition, continuous concentration of particulate matter under 2.5 µm nominal diameter (PM2.5) was measured indoors and immediately outside the residence. Baseline conditions were established in the first two weeks prior to the PAC being turned on, followed by six weeks with the PAC in operation. Extensive clinical interviews and self-reported measures were administered at baseline, weekly during the study period, and at study completion. To date, nine participants have fully completed the study. The remaining 10-15 participants are expected to be completed by March 2025. Preliminary analysis indicates 100% of participants found the PAC acceptable for use and 78% said they strongly liked having a PAC in their home. Self-perceived improvements in indoor air quality were noted by 88% of participants. Additionally, 80% of the participants completed nearly all of the study measures. While this is primarily a feasibility and acceptability study, preliminary data suggests the PAC did reduce PM2.5 concentrations and had nominal influence on symptom severity as 44% of participants reported improved respiratory health. A challenge noted by the participants was connecting the indoor air quality units to the internet via Bluetooth. Overall, the preliminary results indicate that PAC are feasible and acceptable to Veterans with clinically diagnosed chronic respiratory health conditions and MDD. Given that the pilot data also showed improvement in symptoms, a controlled clinical intervention trial is warranted.

Over the past decade, studies have been conducted to increase the understanding of associations between the fecal microbiome and human health. In conjunction, researchers have investigated the effects of study design, methods, molecular processing, and sequencing techniques. However, a lack of standardization of fecal sample collection methodology has introduced heterogeneity in sequencing results. Sources of variability include sample collection methods, storage temperatures, and transport times. Here we present 16S rRNA gene amplicon sequencing results from two sample collection methods (unstabilized sterile swab and stabilized OmniGene Gut Kits) collected from the same fecal specimens. The paired samples were collected either at the research facility or the participants' home and ground shipped to the research facility at ambient temperature. Therefore, samples were exposed to variable temperatures and transport times. We found that fecal sample collection methods resulted in taxonomic and diversity differences that showed distinct patterns between swab and OmniGene samples. Swab samples were disproportionally affected by increased transport time, but differences in taxa and diversity were driven more by sample collection method, as compared to transport time. Based on previous studies, many of the taxa that were associated with sample collection methods and transport times have clinical relevance. Collectively, this research highlights: 1) the need for further standardization of methods for fecal microbiome studies; 2) limitations of direct comparisons between different fecal sample collection methods; and 3) the importance of careful consideration of sample collection methods for future studies and meta-analyses.

Traumatic brain injuries (TBI) frequently occur and can lead to lasting negative cognitive, physical, and mental health outcomes. The biological response to even mild TBIs (mTBI) includes well-characterized inflammatory sequelae that start immediately post-injury, remain for weeks, and can develop into long-term systemic inflammation. Studies have shown that TBI influences multiple physiological systems, including the gastrointestinal tract, through bidirectional communication modulated, in part, by the gut microbiome. Brainstem functioning post-TBI, as measured by acoustic startle sensorimotor processing, might play a role in this feedback loop. The current study investigated pre- to post-TBI (lateral fluid percussion injury model) changes in microbial communities and acoustic startle response in male and female rats. That is, the influence of mTBI on gut microbiome and sensorimotor processing was explored to examine: 1) overall and sex-specific differences in the gut microbiome and taxa in response to mTBI; 2) overall and sex-specific differences in sensorimotor processing following mTBI; and 3) associations between the gut microbiome and sensorimotor processing. Results showed mTBI had a limited effect on microbial diversity overall, and the same was observed in males and females independently. Yet, mTBI was associated with differences in 13 genus-level taxa. Further evaluation highlighted that 11 of the 13 genus-level taxa were sex-specific, with several being known to have short-chain fatty acid-producing capabilities. Alterations in sensorimotor processing were identified following mTBI; however, no sex-specific differences were evident. In addition, no associations were observed between sensorimotor processing and the gut microbiome. This study contributes longitudinal and sex-specific findings to the growing body of research examining the diverse effects of mTBI on the brain and gut microbial communities.

BACKGROUND:Burn injury produces a complex biological response across multiple organs and biological systems. Nonetheless, current understanding regarding the neurologic response to burn injury is limited. Research suggests that disruption of the blood-brain barrier may play a role in central nervous system (CNS) damage after burn trauma. As such, the purpose of this study was to investigate systemic circulating biomarkers, frequently associated with neuronal injury, to gain an understanding of their relationship to burn injury severity. METHODS:Blood from 56 patients admitted to the burn intensive care units was taken within 24 hours and analyzed for four CNS-related biomarkers in plasma (i.e., ubiquitin C-terminal hydrolase L1, tau protein, glial fibrillary acidic protein, and neurofilament light). Clinical information regarding demographics, burn severity, and health outcomes was also obtained. RESULTS:We observed that increased burn severity, as measured by total burn surface area (TBSA), was significantly associated with increased ubiquitin C-terminal hydrolase L1, neurofilament light, and tau. Glial fibrillary acidic protein was not associated with burn severity. In a predictive model of days spent in the hospital after injury, the accuracy of the four CNS-related biomarkers was only improved by 1% when TBSA was included (i.e., 38.3% accuracy with only biomarkers vs. 39.4% accuracy with biomarkers and TBSA). CONCLUSIONS:Overall, findings from this novel study highlight an association between burn injury severity and CNS-related biomarkers, thereby providing a foundation for future studies to explore both potential mechanisms associated with burn-related neurologic damage and associated functional impairments.

Obesity, associated with the intake of a high-fat diet (HFD), and anxiety are common among those living in modern urban societies. Recent studies suggest a role of microbiome-gut-brain axis signaling, including a role for brain serotonergic systems in the relationship between HFD and anxiety. Evidence suggests the gut microbiome and the serotonergic brain system together may play an important role in this response. Here we conducted a nine-week HFD protocol in male rats, followed by an analysis of the gut microbiome diversity and community composition, brainstem serotonergic gene expression (tph2, htr1a, and slc6a4), and anxiety-related defensive behavioral responses. We show that HFD intake decreased alpha diversity and altered the community composition of the gut microbiome in association with obesity, increased brainstem tph2, htr1a and slc6a4 mRNA expression, including in the caudal part of the dorsomedial dorsal raphe nucleus (cDRD), a subregion previously associated with stress- and anxiety-related behavioral responses, and, finally, increased anxiety-related defensive behavioral responses. The HFD increased the Firmicutes/Bacteroidetes ratio relative to control diet, as well as higher relative abundances of Blautia, and decreases in Prevotella. We found that tph2, htr1a and slc6a4 mRNA expression were increased in subregions of the dorsal raphe nucleus in the HFD, relative to control diet. Specific bacterial taxa were associated with increased serotonergic gene expression in the cDRD. Thus, we propose that HFD-induced obesity is associated with altered microbiome-gut-serotonergic brain axis signaling, leading to increased anxiety-related defensive behavioral responses in rats.

Recent microbiome research has incorporated a higher number of samples through more participants in a study, longitudinal studies, and metanalysis between studies. Physical limitations in a sequencing machine can result in samples spread across sequencing runs. Here we present the results of sequencing nearly 1000 16S rRNA gene sequences in fecal (stabilized and swab) and oral (swab) samples from multiple human microbiome studies and positive controls that were conducted with identical standard operating procedures. Sequencing was performed in the same center across 18 different runs. The simplified mock community showed limitations in accuracy, while precision (e.g., technical variation) was robust for the mock community and actual human positive control samples. Technical variation was the lowest for stabilized fecal samples, followed by fecal swab samples, and then oral swab samples. The order of technical variation stability was inverse of DNA concentrations (e.g., highest in stabilized fecal samples), highlighting the importance of DNA concentration in reproducibility and urging caution when analyzing low biomass samples. Coefficients of variation at the genus level also followed the same trend for lower variation with higher DNA concentrations. Technical variation across both sample types and the two human sampling locations was significantly less than the observed biological variation. Overall, this research providing comparisons between technical and biological variation, highlights the importance of using positive controls, and provides semi-quantified data to better understand variation introduced by sequencing runs. KEY POINTS: • Mock community and positive control accuracy were lower than precision. • Samples with lower DNA concentration had increased technical variation across sequencing runs. • Biological variation was significantly higher than technical variation due to sequencing runs.

Objective: The purpose of this study was to evaluate the influence of a new course of antidepressant monotherapy on gut and oral microbiomes and the relationship to depressive symptoms. Methods: Longitudinal microbiome samples obtained from 10 U.S. veterans were analyzed. Baseline samples were taken before a new course of antidepressant monotherapy (either switching from a previous treatment or starting a new treatment). Targeted genomic sequencing of the micro- biome samples was used to analyze changes in taxonomy and diversity across participants, medications, and medication class. Associations between these changes and Patient Health Questionnaire-9 (PHQ-9) scores were analyzed. Results: Taxonomic variability was observed across participants, with the individual being the main microbial community driver. In terms of the fecal microbiome, antidepressants were associated with shifts toward Bacteroides being less abundant and Bfautia, , Pseudomonas, , or Faecafibacterium being more abundant. Likewise, the composition of the oral microbiome was variable, with individual participants being the primary drivers of community composition. In the oral samples, the relative abundance of Haemophifus decreased after antidepressants were started. Increases in Bfautia and decreases in Bacteroides were associated with lower PHQ-9 scores. Conclusions: Antidepressants were found to influence fecal and oral microbiomes such that a new course of antidepressant monotherapy was associated with taxonomic alterations toward healthier states in both fecal and oral microbiomes, which were associated with decreases in depressive symptoms. Additional longitudinal research is required to increase understanding of microbiomes and symptom-based changes, with a particular focus on potential differences between medication classes and underlying mechanisms.

Previous studies have shown that the in vivo administration of soil-derived bacteria with anti-inflammatory and immunoregulatory properties, such as Mycobacterium vaccae NCTC 11659, can prevent a stress-induced shift toward an inflammatory M1 microglial immunophenotype and microglial priming in the central nervous system (CNS). It remains unclear whether M. vaccae NCTC 11659 can act directly on microglia to mediate these effects. This study was designed to determine the effects of M. vaccae NCTC 11659 on the polarization of naïve BV-2 cells, a murine microglial cell line, and BV-2 cells subsequently challenged with lipopolysaccharide (LPS). Briefly, murine BV-2 cells were exposed to 100 µg/mL whole-cell, heat-killed M. vaccae NCTC 11659 or sterile borate-buffered saline (BBS) vehicle, followed, 24 h later, by exposure to 0.250 µg/mL LPS (Escherichia coli 0111: B4; n = 3) in cell culture media vehicle (CMV) or a CMV control condition. Twenty-four hours after the LPS or CMV challenge, cells were harvested to isolate total RNA. An analysis using the NanoString platform revealed that, by itself, M. vaccae NCTC 11659 had an “adjuvant-like” effect, while exposure to LPS increased the expression of mRNAs encoding proinflammatory cytokines, chemokine ligands, the C3 component of complement, and components of inflammasome signaling such as Nlrp3. Among LPS-challenged cells, M. vaccae NCTC 11659 had limited effects on differential gene expression using a threshold of 1.5-fold change. A subset of genes was assessed using real-time reverse transcription polymerase chain reaction (real-time RT-PCR), including Arg1, Ccl2, Il1b, Il6, Nlrp3, and Tnf. Based on the analysis using real-time RT-PCR, M. vaccae NCTC 11659 by itself again induced “adjuvant-like” effects, increasing the expression of Il1b, Il6, and Tnf while decreasing the expression of Arg1. LPS by itself increased the expression of Ccl2, Il1b, Il6, Nlrp3, and Tnf while decreasing the expression of Arg1. Among LPS-challenged cells, M. vaccae NCTC 11659 enhanced LPS-induced increases in the expression of Nlrp3 and Tnf, consistent with microglial priming. In contrast, among LPS-challenged cells, although M. vaccae NCTC 11659 did not fully prevent the effects of LPS relative to vehicle-treated control conditions, it increased Arg1 mRNA expression, suggesting that M. vaccae NCTC 11659 induces an atypical microglial phenotype. Thus, M. vaccae NCTC 11659 acutely (within 48 h) induced immune-activating and microglial-priming effects when applied directly to murine BV-2 microglial cells, in contrast to its long-term anti-inflammatory and immunoregulatory effects observed on the CNS when whole-cell, heat-killed preparations of M. vaccae NCTC 11659 were given peripherally in vivo.

While many studies of intestinal permeability (IP) are focused on those with gastrointestinal (GI) disorders, there is a rising trend to analyze IP among individuals with mental health conditions including posttraumatic stress disorder (PTSD) with and without diagnosed GI conditions. This interest stems from the association between gut dysbiosis and chronic inflammation, which are mechanisms linked to stress-related somatic and mental health conditions. Efforts to date have resulted in the exploration of non-invasive and feasible measures to identify an IP biomarker that could also serve as a treatment target. Additionally, those conducting studies regarding IP often recruit individuals without health problems and compare levels of biomarkers of IP to those obtained from participants with conditions of interest. This study aimed to assess correlations between blood-based biomarkers of IP, as well as examine the association between blood-based biomarkers of IP and PTSD symptoms. Blood was sampled from seventeen United States military Veterans with variable severity of PTSD symptoms per the posttraumatic checklist for DSM-5 (PCL-5) (n = 6 with scores over 31 indicating clinically meaningful symptoms of PTSD; overall range 0-49, mean 20.8, standard deviation 15.7) and analyzed blood biomarkers of IP including citrulline, diamine oxidase, glucagon-like peptide-2, intestinal fatty-acid binding protein, lipopolysaccharide binding protein, lipopolysaccharide, and zonulin. Correlations between the IP blood-based biomarkers ranged from rho of-0.31 to 0.35. None of the measured biomarkers were significantly correlated to PTSD symptom severity scores (rho of-0.34 to 0.05). Although based on limited sample size, our results call into question the specificity of blood-based biomarkers of IP when: (1) studying persons with and without PTSD symptoms in whom clinical GI disorders are not necessarily the focus of the study; and (2) comparing IP results among individuals with well-defined disease states to those without the disease (e.g., controls). Further studies are needed to explore the role of external factors (e.g., nutrition, obesity, alcohol use) on IP and to determine if the biomarkers studied are appropriate for measuring IP in people with a range of symptoms related to PTSD.

Compared to microbiomes on other skin sites, the bacterial microbiome of the human hand has been found to have greater variability across time. To increase understanding regarding the longitudinal transfer of the hand microbiome to objects in the built environment, and vice versa, 22 participants provided skin microbiome samples from their dominant hands, as well as from frequently and infrequently touched objects in their office environments. Additional longitudinal samples from home environments were obtained from a subset of 11 participants. We observed stability of the microbiomes of both the hand and built environments within the office and home settings; however, differences in the microbial communities were detected across the two built environments. Occupants' frequency of touching an object correlated to that object having a higher relative abundance of human microbes, yet the percent of shared microbes was variable by participants. Finally, objects that were horizontal surfaces in the built environment had higher microbial diversity as compared to objects and the occupants' hands. This study adds to the existing knowledge of microbiomes of the built environment, enables more detailed studies of indoor microbial transfer, and contributes to future models and building interventions to reduce negative outcomes and improve health and well-being.

ABSTRACT Social and economic inequities can have a profound impact on human health, particularly on the development and progression of chronic disease. For military veterans, exposure to unique environments and circumstances may further impact their health. There continues to be limited work regarding the influence of mental health within the context of socioeconomic inequities. In this cross-sectional study, we hypothesized that veterans residing in food deserts (e.g., places in which there is a lack of access to sufficient and/or nutritious food) would have decreased gut microbial species (α-diversity), different microbiome community compositions, and poorer quality of diet and mental health compared to non-food desert residents. The fecal microbiome of 342 military veterans was sequenced, and microbiome diversity and community composition were evaluated. Although dietary quality and α-diversity did not significantly differ by food desert status, resident status (food desert versus non-food desert) accounted for a moderate influence on β-diversity (2.4%). Factors such as race and psychiatric diagnoses accounted for greater proportions of β-diversity influence (7% and 10%, respectively). Moreover, more participants with current post-traumatic stress disorder lived in food deserts ( P < 0.04), and there were significantly more participants in the non-food desert group diagnosed with substance use disorders ( P = 0.002) and current alcohol use disorder ( P = 0.04). These findings suggest that living in a food desert, in combination with additional associated risk factors, may influence gut microbial diversity and composition. To increase ecological validity, researchers investigating the influence of inter-related biopsychosocial factors over time may benefit from adopting a life-course perspective. IMPORTANCE Social and economic inequities can have a profound impact on human health. The inequities could result in alterations to the gut microbiome, an important factor that may have profound abilities to alter health outcomes. Moreover, the strong correlations between social and economic inequities have been long understood. However, to date, limited research regarding the microbiome and mental health within the context of socioeconomic inequities exists. One particular inequity that may influence both mental health and the gut microbiome is living in a food desert. Persons living in food deserts may lack access to sufficient and/or nutritious food and often experience other inequities, such as increased exposure to air pollution and poor access to healthcare. Together, these factors may confer a unique risk for microbial perturbation. Indeed, external factors beyond a food desert might compound over time to have a lasting effect on an individual’s gut microbiome. Therefore, adoption of a life-course approach is expected to increase the ecological validity of research related to social inequities, the gut microbiome, and physical and mental health.

Background and Objectives:Posttraumatic stress disorder (PTSD) and traumatic brain injury (TBI) are associated with chronic inflammation, as inferred from increased, but variable, peripheral levels of cytokines. We sought proof of concept for the notion that peripheral cytokine binding proteins and/or soluble receptors can confound measures of cytokines in those with a history of physical and psychological traumatic exposures. Efforts were focused on one of the major cytokines involved in inflammation, tumor necrosis factor-α (TNF-α). Methods:We examined blood plasma concentrations of TNF-α, its soluble receptors (TNF-soluble receptors (sR) I and TNFsRII), and C-reactive protein (CRP-1) in a cohort of US Veterans. In a previous study, CRP-1 was shown to be reduced by probiotic anti-inflammatory treatment in this patient cohort. All participants (n = 22) were diagnosed with PTSD and had a history of mild TBI with persistent post-concussive symptoms. Exclusion criteria included medications directly targeting inflammation. Results:Molar concentrations of soluble TNFsRI and II exceeded concentrations of the TNF-α ligand. TNFsRI, but not TNFsRII, was significantly associated with CRP-1 (Spearman Rho correlations = 0.518; p=.016 and 0.365; p = .104, respectively). Conclusions:TNF soluble receptors may bind to and sequester free TNF-α, suggesting that only measuring ligand concentrations may not provide a fully comprehensive view of inflammation, and potentially lead to inaccurate conclusions. TNFsRI concentration may provide a better estimate of inflammation than TNF-α for those with PTSD and post-acute mTBI with post-concussive symptoms, a hypothesis that invites further testing in larger studies.

Military Veterans account for 8% of homeless individuals living in the United States. To highlight associations between history of homelessness and the gut microbiome, we compared the gut microbiome of Veterans who reported having a previous experience of homelessness to those from individuals who reported never having experienced a period of homelessness. Moreover, we examined the impact of the cumulative exposure of prior and current homelessness to understand possible associations between these experiences and the gut microbiome. Microbiome samples underwent genomic sequencing and were analyzed based on alpha diversity, beta diversity, and taxonomic differences. Additionally, demographic information, dietary data, and mental health history were collected. A lifetime history of homelessness was found to be associated with alcohol use disorder, substance use disorder, and healthy eating index compared to those without such a history. In terms of differences in gut microbiota, beta diversity was significantly different between Veterans that had experienced homelessness and Veterans that had never been homeless ( p = 0.047, Weighted UniFrac), while alpha diversity was similar. The microbial community differences were, in part, driven by a lower relative abundance of Akkermansia in Veterans that had experienced homelessness (mean ± SD; 1.07 ± 3.85) compared to Veterans that had never been homeless (2.02 ± 5.36) ( p = 0.014, ancom-bc2). Additional research is required to facilitate understanding regarding complex associations between homelessness, the gut microbiome, and mental and physical health conditions, with a focus on increasing understanding regarding the longitudinal impact of housing instability throughout the lifespan. Importance Although there are known stressors related to homelessness, as well as chronic health conditions experienced by those without stable housing, there has been limited work evaluating the associations between microbial community composition and homelessness. We analyzed, for the first time, bacterial gut microbiome associations among those with experiences of homelessness on alpha diversity, beta diversity, and taxonomic differences. Additionally, we characterized the influences of diet, demographic characteristics, military service history and mental health conditions on the microbiome of Veterans with and without any lifetime history of homelessness. Future longitudinal research to evaluate the complex relationships between homelessness, the gut microbiome, and mental health outcomes is recommended. Ultimately, differences in the gut microbiome of individuals experiencing and not experiencing homelessness could assist in identification of treatment targets to improve health outcomes.